The present invention relates to the field of optical spectroscopy and in particular without limitation to optical spectroscopy utilizing multivariate optical means for spectral analysis.
Spectroscopic techniques are widely used for determination of the composition of a substance. By spectrally analyzing an optical signal, i.e. a spectroscopic optical signal, the concentration of a particular compound or analyte of the substance can be precisely determined. The concentration of a particular analyte or compound is typically given by an amplitude of a principal component of an optical signal.
U.S. Pat. No. 6,198,531 B1 discloses an embodiment of an optical analysis system for determining an amplitude of a principal component of an optical signal. The known optical analysis system is part of a spectroscopic analysis system suited for, e.g., analyzing which compounds are comprised at which concentrations in a sample. It is well known that light interacting with the sample carries away information about the compounds and their concentrations. The underlying physical processes are exploited in optical spectroscopic techniques in which light of a light source such as, e.g., a laser, a lamp or light emitting diode is directed to the sample for generating an optical signal that carries this information.
For example, light may be absorbed by the sample. Alternatively or in addition, light of a known wavelength may interact with the sample and thereby generate light at a different wavelength due to, e.g. a Raman process. The transmitted and/or generated light then constitutes the optical signal which may also be referred to as the spectrum. The relative intensity of the optical signal as a function of the wavelength is then indicative of the compounds comprised in the sample and their concentrations.
To identify the compounds comprised in the sample and to determine their concentrations the optical signal has to be analyzed. In the known optical analysis system the optical signal is analyzed by dedicated hardware comprising an optical filter. This optical filter has a transmission which depends on the wavelength, i.e. it is designed to weight the optical signal by a spectral weighting function which is given by the wavelength-dependent transmission. The spectral weighting function is chosen such that the total intensity of the weighted optical signal, i.e. of the light transmitted by the filter, is directly proportional to the concentration of a particular compound. Such an optical filter is also denoted a multivariate optical element (MOE), that may be implemented in transmission or reflection geometry. This intensity can then be conveniently detected by a detector such as, e.g., a photodiode. For every compound a dedicated optical filter with a characteristic spectral weighting function is used. The optical filter may be, e.g., an interference filter having a transmission constituting the desired weighting function.
Typically, the principal component comprises a positive part and a negative part. Therefore, a part of the optical signal is directed to a first filter which weights the optical signal by a first spectral weighting function corresponding to the positive part of the principal component, and a further part of the optical signal is directed to a second filter which weights the optical signal by a second spectral weighting function corresponding to the negative part of the principal component. The light transmitted by the first and second filters is then separately detected by a first and a second detector, respectively. The two signals obtained by the two detectors are then subtracted, resulting in a signal with an amplitude corresponding to the concentration of a dedicated compound of the sample.
In this way, instead of the entire spectrum, only a single signal that is proportional to a specific compound of the sample is detected. Hence, a rather expensive charge coupled device (CCD) camera can be effectively replaced by low-cost light sensitive detectors, such as e.g. semiconductor-based photodiodes. However, by means of such a multivariate optical analysis only two signals, namely the positive and the negative parts of the principal component, are obtained. They are representative of the spectroscopic signal itself and also correspond to a non-negligible background signal. For a precise and reliable determination of the concentration of a compound or analyte of the investigated substance, the entire spectroscopic system has to be correctly calibrated. Hence, the correlation between the detected principal component and a corresponding concentration has to be scaled properly.
In many spectroscopic analysis systems elastically scattered radiation as well as dark current of the detector may give rise to appreciable background signals that are superimposed on the intrinsic spectroscopic signal. Typically, spectroscopic signals that have to be analyzed feature relatively narrow peaks in the spectrum compared to the broadband fluorescence or dark current background. Generally, a reliable and sufficient spectroscopic analysis requires effective elimination of broadband background signals.
This can for example be provided by filtering of slowly varying signal components of a spectrum. However, by making use of MOEs only a single signal rather than the entire spectrum is detected. Consequently, a filtering of slowly varying spectral components cannot be performed in a straightforward way. However, background compensation is a necessary step of spectroscopic signal analysis and it also has to be applied on spectroscopic analysis based on multivariate optical analysis.
The advantages of a background compensation scheme are obvious, when for example the background is subject to modifications that might easily occur in the framework of spectroscopic analysis of biological tissue. In particular, when spectroscopic analysis is applied to a variety of different biological tissues featuring different optical properties, a fluorescence background may strongly depend on the type of the biological tissue. Moreover, other effects like scattering of light in a light guiding arrangement providing transmission of collected optical signals to a spectroscopic analysis system may also have a major impact on the background level. Also, when the background is non uniform, i.e. the fluorescence or dark current is not uniform over a large spectral range, subtracting a constant fluorescence and dark current background would falsify the spectroscopic signal to a large extent.